Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 81
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(11): 1000-1007, 2019 Nov.
Artigo em Chinês | MEDLINE | ID: mdl-31878996

RESUMO

Objective To screen a standard homogenous cDNA library of human gastric mucosal epithelium with yeast two-hybrid system and find the proteins that interact with Src-homology 2-containing inositol 5-phosphatase 2 (SHIP2). Methods Using the yeast two-hybrid system, P1 (SH2+5-Ptase) and P2 (PRD+SAM) segments of SHIP2 were used as bait proteins to screen the proteins that bind to SHIP2 from the homogenous cDNA library of human gastric mucosal epithelium. The selected interacting proteins of SHIP2 were verified by co-immunoprecipitation assay. Results A total of 39 positive clones were selected and sequenced for alignment analysis. It was verified that PHB interacted with SHIP2 by reductive hybridization and co-immunoprecipitation assays. Conclusion PHB, the interacting protein of SHIP2 was screened by yeast two-hybrid system from the homogenous cDNA library of human gastric mucosal epithelium.


Assuntos
Epitélio/metabolismo , Mucosa Gástrica/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Proteínas Repressoras/metabolismo , Biblioteca Gênica , Humanos , Inositol Polifosfato 5-Fosfatases , Técnicas do Sistema de Duplo-Híbrido
2.
Biomed Pharmacother ; 118: 109392, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31545285

RESUMO

Laryngeal squamous cell carcinoma (LSCC) is the major type of laryngeal carcinoma. SHIP2 plays a critical role in malignant tumors and is associated with activation of PI3K/Akt signaling pathway. Here, we aimed to explore the impacts of SHIP2 on LSCC Hep-2 cells and the relationship between SHIP2 and radiotherapy. SHIP2 knockdown impairs cell proliferation, invasion, migration and promotes cell apoptosis in this study, suggesting the oncogenic role of SHIP2 in laryngeal cancer. Radiation not only has the similar effect on laryngeal cancer as SHIP2 knockdown, but also causes significant cell cycle G2 arrest, all of which can be significantly enhanced by SHIP2 knockdown. This enhancement effect cause by SHIP2 knockdown derive from the inactivation of PI3K/Akt signaling pathway along with its downstream proteins. Our finding revealed a novel mechanism for sensitivity to radiotherapy caused by SHIP2 knockdown that called descending-SHIP2-mediated radiosensitivity enhancement (DSMRSE).


Assuntos
Carcinoma de Células Escamosas/metabolismo , Neoplasias Laríngeas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Tolerância a Radiação , Apoptose , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Movimento Celular , Humanos , Neoplasias Laríngeas/patologia , Invasividade Neoplásica , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais
3.
Infect Immun ; 87(11)2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31481407

RESUMO

Periodontal disease is a significant health burden, causing tooth loss and poor oral and overall systemic health. Dysbiosis of the oral biofilm and a dysfunctional immune response drive chronic inflammation, causing destruction of soft tissue and alveolar bone supporting the teeth. Treponema denticola, a spirochete abundant in the plaque biofilm of patients with severe periodontal disease, perturbs neutrophil function by modulating appropriate phosphoinositide (PIP) signaling. Through a series of immunoblotting and quantitative PCR (qPCR) experiments, we show that Msp does not alter the gene transcription or protein content of key enzymes responsible for PIP3 signaling: 3' phosphatase and tensin homolog (PTEN), phosphatidylinositol 3-kinase (PI3K), or 5' Src homology 2 domain-containing inositol phosphatase 1 (SHIP1). Instead, using immunoblotting and enzyme-linked immunosorbent assays (ELISAs), we found that Msp activates PTEN through dephosphorylation specifically at the S380 site. Msp in intact organisms or outer membrane vesicles also restricts PIP signaling. SHIP1 phosphatase release was assessed using chemical inhibition and immunoprecipitation to show that Msp moderately decreases SHIP1 activity. Msp also prevents secondary activation of the PTEN/PI3K response. We speculate that this result is due to the redirection of the PIP3 substrate away from SHIP1 to PTEN. Immunofluorescence microscopy revealed a redistribution of PTEN from the cytoplasm to the plasma membrane following exposure to Msp, which may contribute to PTEN activation. Mechanisms of how T. denticola modulates and evades the host immune response are still poorly described, and here we provide further mechanistic evidence of how spirochetes modify PIP signaling to dampen neutrophil function. Understanding how oral bacteria evade the immune response to perpetuate the cycle of inflammation and infection is critical for combating periodontal disease to improve overall health outcomes.


Assuntos
Proteínas de Bactérias/farmacologia , Neutrófilos/efeitos dos fármacos , Fosfatidilinositóis/metabolismo , Porinas/farmacologia , Treponema denticola/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Quimiotaxia , Regulação da Expressão Gênica/efeitos dos fármacos , Imunoprecipitação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Porinas/metabolismo
4.
Int J Mol Sci ; 20(11)2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31212584

RESUMO

Insulin signaling is mediated by a highly integrated network that controls glucose metabolism, protein synthesis, cell growth, and differentiation. Our previous work indicates that the insulin receptor tyrosine kinase substrate (IRTKS), also known as BAI1-associated protein 2-like 1 (BAIAP2L1), is a novel regulator of insulin network, but the mechanism has not been fully studied. In this work we reveal that IRTKS co-localizes with Src homology (SH2) containing inositol polyphosphate 5-phosphatase-2 (SHIP2), and the SH3 domain of IRTKS directly binds to SHIP2's catalytic domain INPP5c. IRTKS suppresses SHIP2 phosphatase to convert phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P3, PIP3) to phosphatidylinositol (3,4) bisphosphate (PI(3,4)P2). IRTKS-knockout significantly increases PI(3,4)P2 level and decreases cellular PI(3,4,5)P3 content. Interestingly, the interaction between IRTKS and SHIP2 is dynamically regulated by insulin, which feeds back and affects the tyrosine phosphorylation of IRTKS. Furthermore, IRTKS overexpression elevates PIP3, activates the AKT-mTOR signaling pathway, and increases cell proliferation. Thereby, IRTKS not only associates with insulin receptors to activate PI3K but also interacts with SHIP2 to suppress its activity, leading to PIP3 accumulation and the activation of the AKT-mTOR signaling pathway to modulate cell proliferation.


Assuntos
Proteínas dos Microfilamentos/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Transdução de Sinais/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Células HEK293 , Células Hep G2 , Humanos , Imunoprecipitação , Insulina/metabolismo , Proteínas dos Microfilamentos/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética , Monoéster Fosfórico Hidrolases/genética , Fosforilação/genética , Fosforilação/fisiologia , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
5.
J Chem Theory Comput ; 15(8): 4318-4331, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31241940

RESUMO

The relative prevalence of native protein-protein interactions (PPIs) are the cornerstone for understanding the structure, dynamics and mechanisms of function of protein complexes. In this study, we develop a scheme for scaling the protein-water interaction in the CHARMM36 force field, in order to better fit the solvation free energy of amino acids side-chain analogues. We find that the molecular dynamics simulation with the scaled force field, CHARMM36s, as well as a recently released version, CHARMM36m, effectively improve on the overly sticky association of proteins, such as ubiquitin. We investigate the formation of a heterodimer protein complex between the SAM domains of the EphA2 receptor and the SHIP2 enzyme by performing a combined total of 48 µs simulations with the different potential functions. While the native SAM heterodimer is only predicted at a low rate of 6.7% with the original CHARMM36 force field, the yield is increased to 16.7% with CHARMM36s, and to 18.3% with CHARMM36m. By analyzing the 25 native SAM complexes formed in the simulations, we find that their formation involves a preorientation guided by Coulomb interactions, consistent with an electrostatic steering mechanism. In 12 cases, the complex could directly transform to the native protein interaction surfaces with only small adjustments in domain orientation. In the other 13 cases, orientational and/or translational adjustments are needed to reach the native complex. Although the tendency for non-native complexes to dissociate has nearly doubled with the modified potential functions, a dissociation followed by a reassociation to the correct complex structure is still rare. Instead, the remaining non-native complexes undergo configurational changes/surface searching, which, however, rarely leads to native structures on a time scale of 250 ns. These observations provide a rich picture of the mechanisms of protein-protein complex formation and suggest that computational predictions of native complex PPIs could be improved further.


Assuntos
Mapas de Interação de Proteínas , Proteínas/metabolismo , Humanos , Simulação de Dinâmica Molecular , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/química , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Proteínas/química , Receptor EphA2/química , Receptor EphA2/metabolismo , Eletricidade Estática , Termodinâmica , Ubiquitina/química , Ubiquitina/metabolismo , Água/metabolismo
6.
Cell Prolif ; 52(5): e12638, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31152465

RESUMO

OBJECTIVES: Terminally differentiated stratified squamous epithelial cells play an important role in barrier protection of the skin. The integrity of epidermal cells is maintained by tight regulation of proliferation and differentiation. The aim of this study was to investigate the role of epigenetic regulator H3K4me3 and its demethylase Jarid1b in the control of epithelial cell differentiation. MATERIALS AND METHODS: RT-qPCR, Western blotting and IHC were used to detect mRNA and protein levels. We analysed cell proliferation by CCK8 assay and cell migration by wound healing assay. ChIP was used to measure H3K4me3 enrichment. A chamber graft model was established for epidermal development. RESULTS: Our studies showed that H3K4me3 was decreased during epidermal differentiation. The H3K4me3 demethylase Jarid1b positively controlled epidermal cell differentiation in vitro and in vivo. Mechanistically, we found that Jarid1b substantially increased the expression of mesenchymal-epithelial transition (MET)-related genes, among which Ovol1 positively regulated differentiation gene expression. In addition, Ovol1 expression was repressed by PI3K-AKT pathway inhibitors and overexpression (O/E) of the PI3K-AKT pathway suppressor Ship1. Knockdown (KD) of Ship1 activated downstream PI3K-AKT pathway and enhanced Ovol1 expression in HaCaT. Importantly, we found that Jarid1b negatively regulated Ship1 expression, but not that of Pten, by directly binding to its promoter to modulate H3K4me3 enrichment. CONCLUSION: Our results identify an essential role of Jarid1b in the regulation of the Ship1/AKT/Ovol1 pathway to promote epithelial cell differentiation.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Proteínas Nucleares/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Proteínas de Ligação a DNA/genética , Células Epidérmicas/citologia , Células Epidérmicas/metabolismo , Transição Epitelial-Mesenquimal , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Histona Desmetilases com o Domínio Jumonji/genética , Masculino , Camundongos , Camundongos Nus , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/antagonistas & inibidores , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética , Regiões Promotoras Genéticas , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Transdução de Sinais , Fatores de Transcrição/genética
7.
Nat Commun ; 10(1): 2157, 2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31089138

RESUMO

T cell senescence and exhaustion are major barriers to successful cancer immunotherapy. Here we show that miR-155 increases CD8+ T cell antitumor function by restraining T cell senescence and functional exhaustion through epigenetic silencing of drivers of terminal differentiation. miR-155 enhances Polycomb repressor complex 2 (PRC2) activity indirectly by promoting the expression of the PRC2-associated factor Phf19 through downregulation of the Akt inhibitor, Ship1. Phf19 orchestrates a transcriptional program extensively shared with miR-155 to restrain T cell senescence and sustain CD8+ T cell antitumor responses. These effects rely on Phf19 histone-binding capacity, which is critical for the recruitment of PRC2 to the target chromatin. These findings establish the miR-155-Phf19-PRC2 as a pivotal axis regulating CD8+ T cell differentiation, thereby paving new ways for potentiating cancer immunotherapy through epigenetic reprogramming of CD8+ T cell fate.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Melanoma Experimental/imunologia , MicroRNAs/metabolismo , Neoplasias Cutâneas/imunologia , Fatores de Transcrição/metabolismo , Transferência Adotiva/métodos , Animais , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/transplante , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Senescência Celular/genética , Senescência Celular/imunologia , Epigênese Genética/imunologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma Experimental/genética , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Complexo Repressor Polycomb 2/imunologia , Complexo Repressor Polycomb 2/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/terapia , Fatores de Transcrição/genética , Fatores de Transcrição/imunologia
8.
Immunol Lett ; 210: 40-46, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31004680

RESUMO

IgE-mediated activation of basophil granulocytes and mast cells follows a bell-shaped dose-response curve. The decreased activation at supraoptimal allergen stimulation is thought to be associated with SH2-containing inositol-5'-phosphatase 1 (SHIP-1). SHIP-1 phosphorylation is inversely related to IgE-mediated releasability of basophils. This study sought to clarify the regulatory role of SHIP-1 in degranulation of basophil granulocytes and mast cells by selective inhibition of the phosphatase function of SHIP-1with 3-α-aminocholestane (3-α-AC). Six grass pollen allergic patients, six non-responder patients and six cultured human primary mast cell lines were included. The effect of 3-α-AC (1-60 µM, 30 min, 37 °C) was analyzed at individual suboptimal, optimal and supra-optimal allergen concentrations. The activity, upregulation of CD63, measured at different conditions was compared to evaluate the maximal effect of selective SHIP-1 inhibition. Basophils of five non-responder patients were treated with 3-α-AC (10 µM, 30 min, 37 °C). At high concentrations (>60 µM) of 3-α-AC, cells appeared to enter apoptosis. The median reactivity increased from 27.1% to 44.9% CD63+ basophils at 10 µM of 3-α-AC and suboptimal allergen stimulation (p = 0.0153). There was no effect on blood basophils of 3-α-AC at optimal or supra-optimal allergen concentrations. In contrast, treatment with more than 6 µM 3-α-AC significantly inhibited mast cell reactivity. 10 µM 3-α-AC reduced median reactivity from 32.85% to 16.5% CD63+ mast cells (p = 0.0465). Treatment with 3-α-AC did not increase response of basophils of non-responder patients. Modulating blood basophils with 3-α-AC enhanced reactivity only at suboptimal allergen concentration, and basophils from non-responders did not regain responsiveness to IgE stimulation. 3-α-AC inhibited the IgE response of mast cells in a dose dependent manner.


Assuntos
Basófilos/imunologia , Basófilos/metabolismo , Imunoglobulina E/imunologia , Mastócitos/imunologia , Mastócitos/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Adulto , Alérgenos/imunologia , Feminino , Humanos , Hipersensibilidade/imunologia , Hipersensibilidade/metabolismo , Masculino , Pessoa de Meia-Idade , Receptores de IgE/metabolismo , Adulto Jovem
9.
Oncol Rep ; 41(4): 2337-2350, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30720128

RESUMO

Src homology 2­containing inositol­5'­phosphatase 1 (SHIP1) serves a vital role in the occurrence and development of hematological tumors, but there is limited knowledge regarding the role of SHIP1 in various solid tumors, including lung cancer. In the present study, the aim was to investigate the expression and functional mechanisms of SHIP1 in non­small cell lung cancer (NSCLC). The Gene Expression Omnibus database demonstrated that SHIP1 had low expression in NSCLC. Further studies using fresh tissues and cell lines also confirmed this observation. Biological function analyses revealed that SHIP1 overexpression notably suppressed cell growth, migration and invasion in vitro and in vivo in NSCLC. Mechanistic analyses indicated that SHIP1 inactivated the phosphoinositide 3­kinase (PI3K)/AKT pathway to suppress signals associated with the cell cycle and epithelial­mesenchymal transition. In clinical specimens, reduced SHIP1 is an unfavorable factor and is negatively associated with the T classification, N classification and clinical stage. Furthermore, patients with low SHIP1 levels exhibited reduced survival rate, compared with patients with high levels of the protein. Notably, the promoter of the SHIP1 gene lacks CpG islands, and the suppression of SHIP1 expression is not associated with epidermal growth factor receptor or Kirsten rat sarcoma mutations. Thus, the present study demonstrated that SHIP1 inhibits cell growth, migration and invasion in NSCLC through the PI3K/AKT pathway. Additionally, reduced SHIP1 expression may be an unfavorable factor for NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Células A549 , Animais , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Proliferação de Células , Transição Epitelial-Mesenquimal , Feminino , Humanos , Neoplasias Pulmonares/mortalidade , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/metabolismo , Estadiamento de Neoplasias , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Taxa de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Molecules ; 24(3)2019 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-30699999

RESUMO

BACKGROUND: Long-term exposure to chronic stress is thought to be a factor closely correlated with the development of metabolic disorders, such as diabetes mellitus and metabolic syndrome. Xiaoyaosan, a Chinese herbal formula, has been described in many previous studies to exert anxiolytic-like or antidepressant effects in chronically stressed rats. However, few studies have observed the effects of Xiaoyaosan on the metabolic disorders induced by chronic stress. OBJECTIVE: We sought to investigate the effective regulation of Xiaoyaosan on 21-day chronic immobility stress (CIS, which is 3 h of restraint immobilization every day)-induced behavioural performance and metabolic responses and to further explore whether the effects of Xiaoyaosan were related to SHIP2 expression in the liver. METHODS: Sixty male Sprague Dawley rats were randomly divided into a control group, a CIS group, a Xiaoyaosan group and a rosiglitazone group. The latter three groups were subjected to 21 days of CIS to generate the stress model. After 21 days of CIS, the effects of Xiaoyaosan on body weight, food intake, and behaviour in the open field test, the sucrose preference test and the forced swimming test were observed following chronic stress. Plasma insulin, cholesterol (CHOL), triglyceride (TG), low-density lipoprotein (LDL-C) and high-density lipoprotein (HDL-C) concentrations and blood glucose were examined, and the protein and mRNA expression levels of SHIP2, p85 and Akt in the liver were measured using RT-qPCR and immunohistochemical staining. RESULTS: Rats exposed to CIS exhibited depression-like behaviours, decreased levels of plasma insulin, CHOL, LDL-C, TG and HDL-C, and increased blood glucose. Increased SHIP2 expression and reduced Akt, p-Akt and p85 expression were also observed in the liver. Xiaoyaosan exerted antidepressant effects and effectively reversed the changes caused by CIS. CONCLUSIONS: These results suggest that Xiaoyaosan attenuates depression-like behaviours and ameliorates stress-induced abnormal levels of insulin, blood glucose, CHOL, LDL-C and HDL-C in the plasma of stressed rats, which may be associated with the regulation of SHIP2 expression to enhance PI3K/Akt signalling activity in the liver.


Assuntos
Medicamentos de Ervas Chinesas/uso terapêutico , Fígado/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Ansiolíticos/uso terapêutico , Antidepressivos/metabolismo , Comportamento Animal , Glicemia/efeitos dos fármacos , Insulina/sangue , Masculino , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
11.
Eur J Pharmacol ; 851: 69-79, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30753865

RESUMO

Brain-derived neurotrophic factor (BDNF), an essential factor for maintaining brain functions, has been reported to be reduced in various neurological diseases, including Alzheimer's disease and major depression. Therefore, new drugs to increase the BDNF expression need to be developed. Since phosphatidylinositol (3,4,5)-trisphosphate, a membrane signaling molecule produced by phosphoinositide 3 (PI3)-kinase in the BDNF signaling, is a candidate target of SH2 domain-containing inositol 5' phosphatase 2 (SHIP2, a 5'-lipid phosphatase), the present study examined the effect of a SHIP2 inhibitor AS1949490 on Bdnf expression in cultured cortical neurons. BDNF increased its own mRNA levels, and AS1949490 enhanced this positive feedback regulation. The effects of BDNF in combination with AS1949490 on the Bdnf mRNA levels were blocked by inhibitors of mitogen-activated protein kinase kinase (U0126), PI3-kinase (LY294002), phospholipase Cγ (U73122), and protein kinase C (bisindolylmaleimide I), whereas the effect of BDNF alone was inhibited only by U0126. The mRNA stability assay using actinomycin D demonstrated that AS1949490 reduced degradation of the self-amplified Bdnf mRNA levels, and this effect was disappeared in the presence of bisindolylmaleimide I. These results suggest that BDNF promoted the Bdnf mRNA stabilization in a protein kinase C-dependent manner only in the presence of AS1949490, thereby enhancing Bdnf expression. Furthermore, behavioral analyses indicated that central administration of AS1949490 caused memory-improving and anti-depressant effects in passive avoidance test and forced swim test, respectively. Therefore, inhibition of SHIP2 appears to be valuable therapeutic strategy against neurological disorders associated with insufficient BDNF functions.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/genética , Córtex Cerebral/citologia , Neurônios/efeitos dos fármacos , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/antagonistas & inibidores , Proteína Quinase C/metabolismo , Estabilidade de RNA/efeitos dos fármacos , Tiofenos/farmacologia , Peptídeos beta-Amiloides/toxicidade , Animais , Células Cultivadas , Citoproteção/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Memória/efeitos dos fármacos , Camundongos , Neurônios/metabolismo , Fragmentos de Peptídeos/toxicidade , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , Ratos
12.
FASEB J ; 33(2): 2858-2869, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30321069

RESUMO

Metformin, the first-line drug to treat type 2 diabetes (T2D), inhibits mitochondrial glycerolphosphate dehydrogenase in the liver to suppress gluconeogenesis. However, the direct target and the underlying mechanisms by which metformin increases glucose uptake in peripheral tissues remain uncharacterized. Lipid phosphatase Src homology 2 domain-containing inositol-5-phosphatase 2 (SHIP2) is upregulated in diabetic rodent models and suppresses insulin signaling by reducing Akt activation, leading to insulin resistance and diminished glucose uptake. Here, we demonstrate that metformin directly binds to and reduces the catalytic activity of the recombinant SHIP2 phosphatase domain in vitro. Metformin inhibits SHIP2 in cultured cells and in skeletal muscle and kidney of db/db mice. In SHIP2-overexpressing myotubes, metformin ameliorates reduced glucose uptake by slowing down glucose transporter 4 endocytosis. SHIP2 overexpression reduces Akt activity and enhances podocyte apoptosis, and both are restored to normal levels by metformin. SHIP2 activity is elevated in glomeruli of patients with T2D receiving nonmetformin medication, but not in patients receiving metformin, compared with people without diabetes. Furthermore, podocyte loss in kidneys of metformin-treated T2D patients is reduced compared with patients receiving nonmetformin medication. Our data unravel a novel molecular mechanism by which metformin enhances glucose uptake and acts renoprotectively by reducing SHIP2 activity.-Polianskyte-Prause, Z., Tolvanen, T. A., Lindfors, S., Dumont, V., Van, M., Wang, H., Dash, S. N., Berg, M., Naams, J.-B., Hautala, L. C., Nisen, H., Mirtti, T., Groop, P.-H., Wähälä, K., Tienari, J., Lehtonen, S. Metformin increases glucose uptake and acts renoprotectively by reducing SHIP2 activity.


Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Nefropatias/prevenção & controle , Metformina/farmacologia , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/antagonistas & inibidores , Animais , Células Cultivadas , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Humanos , Hipoglicemiantes/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Podócitos/citologia , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Ratos
13.
J Cancer Res Ther ; 14(Supplement): S937-S941, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30539826

RESUMO

Lymph node metastasis is the most common form of metastasis in breast cancer and a crucial indicator influencing breast cancer treatment results. The biological process of lymph node metastasis involves deficiency and mutation of tumor suppressor genes. PTEN and SHIP are critical indicators used to detect occurrence, development, invasion, and metastasis of breast cancer. Loss of expressions of PTEN and SHIP may contribute to lymphatic metastasis of breast cancer, so they can be used as objective indicators for judging the biological behavior of breast cancer. In this study, we perform a comprehensive analysis to investigate the effect of PTEN and SHIP gene expression on regulating lymph node metastasis in breast cancer.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Metástase Linfática/patologia , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Neoplasias da Mama/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Linfonodos/patologia , Metástase Linfática/genética , Domínios de Homologia de src
14.
Nat Commun ; 9(1): 5041, 2018 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-30487552

RESUMO

Apical-basal polarization is essential for epithelial tissue formation, segregating cortical domains to perform distinct physiological functions. Cortical lipid asymmetry has emerged as a determinant of cell polarization. We report a network of phosphatidylinositol phosphate (PIP)-modifying enzymes, some of which are transcriptionally induced upon embedding epithelial cells in extracellular matrix, and that are essential for apical-basal polarization. Unexpectedly, we find that PI(3,4)P2 localization and function is distinct from the basolateral determinant PI(3,4,5)P3. PI(3,4)P2 localizes to the apical surface, and Rab11a-positive apical recycling endosomes. PI(3,4)P2 is produced by the 5-phosphatase SHIP1 and Class-II PI3-Kinases to recruit the endocytic regulatory protein SNX9 to basolateral domains that are being remodeled into apical surfaces. Perturbing PI(3,4)P2 levels results in defective polarization through subcortical retention of apically destined vesicles at apical membrane initiation sites. We conclude that PI(3,4)P2 is a determinant of apical membrane identity.


Assuntos
Fosfatidilinositóis/metabolismo , Animais , Cães , Endossomos/metabolismo , Membranas Intracelulares/metabolismo , Células Madin Darby de Rim Canino , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo
15.
Cell Rep ; 25(5): 1118-1126, 2018 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-30380404

RESUMO

ß-Glucan-induced trained immunity in myeloid cells leads to long-term protection against secondary infections. Although previous studies have characterized this phenomenon, strategies to boost trained immunity remain undefined. We found that ß-glucan-trained macrophages from mice with a myeloid-specific deletion of the phosphatase SHIP-1 (LysMΔSHIP-1) showed enhanced proinflammatory cytokine production in response to lipopolysaccharide. Following ß-glucan training, SHIP-1-deficient macrophages exhibited increased phosphorylation of Akt and mTOR targets, correlating with augmented glycolytic metabolism. Enhanced training in the absence of SHIP-1 relied on histone methylation and acetylation. Trained LysMΔSHIP-1 mice produced increased amounts of proinflammatory cytokines upon rechallenge in vivo and were better protected against Candida albicans infection compared with control littermates. Pharmacological inhibition of SHIP-1 enhanced trained immunity against Candida infection in mouse macrophages and human peripheral blood mononuclear cells. Our data establish proof of concept for improvement of trained immunity and a strategy to achieve it by targeting SHIP-1.


Assuntos
Candidíase/enzimologia , Candidíase/imunologia , Imunidade , Células Mieloides/enzimologia , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , beta-Glucanas/farmacologia , Animais , Candida albicans/fisiologia , Candidíase/microbiologia , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Macrófagos/microbiologia , Camundongos Endogâmicos C57BL , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/antagonistas & inibidores
16.
Sci Rep ; 8(1): 15467, 2018 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-30341350

RESUMO

Antigen (Ag)-mediated crosslinking of IgE-loaded high-affinity receptors for IgE (FcεRI) on mast cells (MCs) triggers activation of proinflammatory effector functions relevant for IgE-associated allergic disorders. The cytosolic tyrosine kinase BTK and the SH2-containing inositol-5'-phosphatase SHIP1 are central positive and negative regulators of Ag-triggered MC activation, respectively, contrarily controlling Ca2+ mobilisation, degranulation, and cytokine production. Using genetic and pharmacological techniques, we examined whether BTK activation in Ship1-/- MCs is mandatory for the manifestation of the well-known hyperactive phenotype of Ship1-/- MCs. We demonstrate the prominence of BTK for the Ship1-/- phenotype in a manner strictly dependent on the strength of the initial Ag stimulus; particular importance for BTK was identified in Ship1-/- bone marrow-derived MCs in response to stimulation with suboptimal Ag concentrations. With respect to MAPK activation, BTK showed particular importance at suboptimal Ag concentrations, allowing for an analogous-to-digital switch resulting in full activation of ERK1/2 already at low Ag concentrations. Our data allow for a more precise definition of the role of BTK in FcεRI-mediated signal transduction and effector function in MCs. Moreover, they suggest that reduced activation or curtate expression of SHIP1 can be compensated by pharmacological inhibition of BTK and vice versa.


Assuntos
Tirosina Quinase da Agamaglobulinemia/metabolismo , Antígenos/metabolismo , Imunoglobulina E/metabolismo , Mastócitos/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/deficiência , Animais , Células da Medula Óssea/citologia , Cálcio/metabolismo , Degranulação Celular , Citocinas/biossíntese , Diglicerídeos/metabolismo , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Mastócitos/fisiologia , Camundongos , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Fosfolipase C gama/metabolismo , Fosforilação , Receptores de IgE/metabolismo , Transdução de Sinais
17.
Sci Signal ; 11(548)2018 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-30228226

RESUMO

Sustained activation of extracellular signal-regulated kinase (ERK) drives pathologies caused by mutations in fibroblast growth factor receptors (FGFRs). We previously identified the inositol phosphatase SHIP2 (also known as INPPL1) as an FGFR-interacting protein and a target of the tyrosine kinase activities of FGFR1, FGFR3, and FGFR4. We report that loss of SHIP2 converted FGF-mediated sustained ERK activation into a transient signal and rescued cell phenotypes triggered by pathologic FGFR-ERK signaling. Mutant forms of SHIP2 lacking phosphoinositide phosphatase activity still associated with FGFRs and did not prevent FGF-induced sustained ERK activation, demonstrating that the adaptor rather than the catalytic activity of SHIP2 was required. SHIP2 recruited Src family kinases to the FGFRs, which promoted FGFR-mediated phosphorylation and assembly of protein complexes that relayed signaling to ERK. SHIP2 interacted with FGFRs, was phosphorylated by active FGFRs, and promoted FGFR-ERK signaling at the level of phosphorylation of the adaptor FRS2 and recruitment of the tyrosine phosphatase PTPN11. Thus, SHIP2 is an essential component of canonical FGF-FGFR signal transduction and a potential therapeutic target in FGFR-related disorders.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Quinases da Família src/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem Celular Tumoral , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/genética , Células HEK293 , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética , Fosforilação , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/genética , Quinases da Família src/genética
18.
Eur J Med Chem ; 157: 405-422, 2018 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-30103190

RESUMO

SH2 domain-containing inositol 5'-phosphatase 2 (SHIP2) is a lipid phosphatase that produce phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2) from phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P3), and is involved in many diseases such as neurodegenerative diseases. A recent report demonstrating that SHIP2 inhibition decreased tau hyperphosphorylation induced by amyloid ß and rescued memory impairment in a transgenic Alzheimer's disease mouse model indicates SHIP2 can be a promising therapeutic target for Alzheimer's disease. In the present study, we have developed novel, potent SHIP2 inhibitors by extensive structural elaboration of crizotinib discovered from a high-throughput screening. Our representative compound 43 potently inhibited SHIP2 activity as well as GSK3ß activation in HT22 neuronal cells. It was also shown that 43 has favorable physicochemical properties, especially high brain penetration. Considering SHIP2 is one of key signal mediators for tau hyperphosphorylation, our potent SHIP2 inhibitor 43 may function as a promising lead compound for the treatment of Alzheimer's disease.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/antagonistas & inibidores , Pirazóis/química , Pirazóis/farmacologia , Piridinas/química , Piridinas/farmacologia , Doença de Alzheimer/enzimologia , Animais , Crizotinibe , Cães , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Estrutura Molecular , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Pirazóis/síntese química , Piridinas/síntese química , Ratos , Relação Estrutura-Atividade , Células Tumorais Cultivadas
19.
Arch Biochem Biophys ; 656: 31-37, 2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30165040

RESUMO

SH2 domain-containing inositol 5-phosphatase 2 (SHIP2) binds with the Y1356-phosphorylated hepatocyte growth factor (HGF) receptor, c-MET, through its SH2 domain, which is essential for the role of SHIP2 in HGF-induced cell scattering and cell spreading. Previously, the experimental structure of the SH2 domain from SHIP2 (SHIP2-SH2) had not been reported, and its interaction with the Y1356-phosphorylated c-MET had not been investigated from a structural point of view. In this study, the solution structure of SHIP2-SH2 was determined by NMR spectroscopy, where it was found to adopt a typical SH2-domain fold that contains a positively-charged pocket for binding to phosphotyrosine (pY). The interaction between SHIP2-SH2 and a pY-containing peptide from c-MET (Y1356 phosphorylated) was investigated through NMR titrations. The results showed that the binding affinity of SHIP2-SH2 with the phosphopeptide is at low micromolar level, and the binding interface consists of the positively-charged pocket and its surrounding regions. Furthermore, R28, S49 and R70 were identified as key residues for the binding and may directly interact with the pY. Taken together, these findings provide structural insights into the binding of SHIP2-SH2 with the Y1356-phosphorylated c-MET, and lay a foundation for further studies of the interactions between SHIP2-SH2 and its various binding partners.


Assuntos
Fragmentos de Peptídeos/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/química , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Fosfotirosina/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Domínios de Homologia de src , Sequência de Aminoácidos , Sítios de Ligação , Humanos , Espectroscopia de Ressonância Magnética , Mutação , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética , Ligação Proteica , Alinhamento de Sequência , Domínios de Homologia de src/genética
20.
J Cell Biol ; 217(10): 3577-3592, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30087126

RESUMO

INPP5K (SKIP) is an inositol 5-phosphatase that localizes in part to the endoplasmic reticulum (ER). We show that recruitment of INPP5K to the ER is mediated by ARL6IP1, which shares features of ER-shaping proteins. Like ARL6IP1, INPP5K is preferentially localized in ER tubules and enriched, relative to other ER resident proteins (Sec61ß, VAPB, and Sac1), in newly formed tubules that grow along microtubule tracks. Depletion of either INPP5K or ARL6IP1 results in the increase of ER sheets. In a convergent but independent study, a screen for mutations affecting the distribution of the ER network in dendrites of the PVD neurons of Caenorhabditis elegans led to the isolation of mutants in CIL-1, which encodes the INPP5K worm orthologue. The mutant phenotype was rescued by expression of wild type, but not of catalytically inactive CIL-1. Our results reveal an unexpected role of an ER localized polyphosphoinositide phosphatase in the fine control of ER network organization.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , Dendritos/enzimologia , Retículo Endoplasmático/enzimologia , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Animais , Células COS , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Dendritos/genética , Retículo Endoplasmático/genética , Deleção de Genes , Células HeLa , Humanos , Camundongos , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA